Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells.

Abstract

Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay, DARPP-32-overexpressing AGS cells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.

• Authors: Shoumin Zhu, Jun Hong, Manish K Tripathi, Vikas Sehdev, Abbes Belkhiri, Wael El-Rifai
• Journal: Molecular cancer research : MCR (Mol Cancer Res) Vol. 11 Issue 1 Pg. 86-94 (Jan 2013) ISSN: 1557-3125 [Electronic] United States
• PMID: 23160836 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: ©2012 AACR.

Chemical References

• CXCR4 protein, human
• Dopamine and cAMP-Regulated Phosphoprotein 32
• PPP1R1B protein, human
• Receptors, CXCR4
• Matrix Metalloproteinase 2
• MMP14 protein, human
• Matrix Metalloproteinase 14

Topics

• Cell Line, Tumor
• Dopamine and cAMP-Regulated Phosphoprotein 32 (biosynthesis, genetics, metabolism)
• HEK293 Cells
• Humans
• Matrix Metalloproteinase 14 (metabolism)
• Matrix Metalloproteinase 2 (metabolism)
• Neoplasm Invasiveness
• Receptors, CXCR4 (biosynthesis, genetics, metabolism)
• Signal Transduction
• Stomach Neoplasms (metabolism, pathology)